A barrier to the successful commercialization of liposomes has been the absence of adequate control over the methods for manufacturing liposomes in large quantities. For regulatory and therapeutic concerns, reproducible products are a necessity. There are numerous published methods for liposome production. Two significant technical concerns govern the commercial usefulness of production methods: content uniformity, and sterility. Hitherto, those production methods that produced liposomes of acceptable content uniformity and sterility had the ancillary problem of size regulation. Previous to the present invention, formulations of preliposome-lyophilate, upon reconstitution, were distributed over a range of diameters larger than 1 micron. This has proven less than ideal for particular therapeutic applications, particularly as to applications benefited from avoidance of the RES.
The use of less than 4% surfactant has been reported (22) to yield stable liposomal preparations. However, the reported procedure required evaporation of toxic organic solvents to prepare a lipid film. Formation of submicron liposomal preparations from such system required sonication. This procedure results in retained organic solvents at levels unsuitable for clinical formulations. Further reconstitution of a thin film presents a material not suitable as a precursor for liposomal preparation due to the substantial difficulty in reconstitution.
Japanese Patent Application No. 91-177731 (Pub. No. 6-183953) (25) discusses a preparation of micelles and what are described as "small vesicles" obtained by treating mixture of phospholipid and nonionic polyoxyethylene surfactant with ultrasonic waves in aqueous solution. The described procedure entails 5% or more of surfactant. No lyophilization of this material was reported.
Japanese Patent Application No. 93-13786 (Pub. No. 6-227966) (26) discusses a thermosensitive liposome preparation for releasing anticancer drug comprising up to 30% nonionic surfactant and phospholipid. This preparation was not disclosed to be lyophilized.
The original liposome preparation of Bangham, et al. (J. Mol. Biol., 1965, 12:238-252) involves dissolving phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to "swell," and the resulting liposomes which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. This technique provides the basis for the development of the small sonicated unilamellar vesicles described by Paphadjopoulos et al. (Biochim. Biophys. Acta., 1968, 135:624-638), and large unilamellar vesicles.
Unilamellar vesicles are produced using an extrusion apparatus by a method described in Cullis et al., PCT Application No. WO 87/00238, published Jan. 16, 1986, entitled "Extrusion Technique for Producing Unilamellar Vesicles" incorporated herein by reference. Vesicles made by this technique, called LUVETS, are extruded under pressure through a membrane filter.
Another class of liposomes are those characterized as having substantially equal lamellar solute distribution. This class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S. Pat. No. 4,522,803 to Lenk, et al., monophasic vesicles as described in U.S. Pat. No. 4,558,579 to Fountain, et al. and frozen and thawed multilamellar vesicles (FATMLV) wherein the vesicles are exposed to at least one freeze and thaw cycle; this procedure is described in Bally et al., PCT Publication No. 87/00043, Jan. 15, 1987, entitled "Multilamellar Liposomes Having Improved Trapping Efficiencies," each of which are incorporated herein by reference. Honda et al. Japanese Patent Pub. No. 60-155109 describes hydrogenated liposomes requiring 5% fatty acid.
Anthracycline antibiotics, namely doxorubicin (Dox), daunorubicin and idarubicin, are among the most effective and widely used anticancer agents. However, their use is limited by acute side effects, mainly acute myelosuppression and chronic cardiotoxicity and natural or acquired drug resistance (1, 2). Liposomes have been extensively studied by different investigators as carriers of this class of compounds. Liposome incorporation has been shown to reduce the cardiotoxicity of Dox in animals (3) and its gastrointestinal side effects and vesicant activity in humans (4). Tumor-targeted formulations of liposomal-Dox are now being evaluated in humans (5). Other interesting studies have shown that multidrug resistance (MDR), a common mechanism of acquired resistance to anthracyclines and other drugs, may be overcome in vitro and in vivo in bone marrow cells by using Dox encapsulated in liposomes containing certain lipids such as cardiolipin or phosphatidylserine (6,7).
The development of pharmaceutical formulations of liposomal-Dox has encountered a great number of technical problems due to the tendency of Dox to leak from the internal aqueous space of the lipid vesicles into the external aqueous milieu. The development of the remote loading technique, which basically consists of keeping the drug protonated inside the liposomes by creating an acidic pH, thus abrogating its ability to cross the liposome membrane, has resulted in formulations with optimal encapsulation efficiencies (8). Still, all liposomal formulations of Dox currently in clinical trials consist of small unilamellar vesicles in suspension and, therefore, particle size stability due to aggregation of particles remains a potential problem.
An alternative approach to enhance the therapeutic index of this class of compounds is the use of new and potentially less toxic and more active analogs. Although a great number of analogs have been synthesized and studied, most of these efforts took place before the phenomenon of MDR was described and well characterized. However, during the last few years, several sub-families of anthracyclines with non cross-resistance properties have been described (9, 10, 11). Because most of them are highly lipophilic and, therefore, not suitable for i.v. administration in water solutions, they require the development of a delivery system for their i.v. administration.
There is a continuing need for improved anticancer drugs, and particularly for improved formulations for delivering anticancer agents to the appropriate sites in a patient's body, while minimizing undesirable side effects.